TITLE: Discovery of small molecule promoters of cardiomyocyte proliferation to restore cardiac performance in disease PROJECT SUMMARY Cardiovascular disease is the leading cause of mortality in developed and developing nations. Existing CVD pharmacotherapy options do not treat any foundational problems in the myocardium and its constituent cardiomyocytes; rather, unloading the heart and reducing vascular risk factors are the primary therapeutic strategies. Future strategies should move beyond prevention and treatment to restoration, regeneration, and replacement of functional cardiac tissue. Two fundamental challenges face the several mechanisms of potential cardiomyocyte renewal (proliferation of endogenous cardiomyocytes, resident progenitor cells, or extracardiac progenitor cells including bone-marrow derived or pluripotent cells): can a sufficient number of new cardiomyocytes be generated to meaningfully improve cardiac function in various diseases? And since most tissues in adult mammals remain post-mitotic to minimize the risk of cellular transformation into an uncontrolled proliferating neoplastic state, can cardiomyocyte-specific proliferation mechanisms be discovered and manipulated? It is unclear that the reported mechanisms of cardiomyocyte proliferation meet both of these challenges. We have recently developed a novel platform to conduct high throughput screening (HTS) on functional human cardiomyocytes matured from iPS cells. Our innovation is a hypothesis-free, phenotypic screening cascade designed to discover previously unknown, cardiomyocyte selective promoters of cardiomyocyte proliferation. Our hypothesis is this approach will ultimately generation drug-like starting points for future disease-modifying cardiovascular therapeutics. The primary HTS assay has been fully optimized in a 384-well format, and as a demonstration of assay readiness, 12,000 compounds have been screened (Z?- factor of 0.4). Multiple hits from pilot screens were identified and were confirmed and validated in concentration response experiments. A battery of downstream assays has been developed to establish a critical path-testing funnel. Several compounds identified from the pilot screen specifically promoted cardiomyocyte proliferation versus fibroblast proliferation, and furthermore promoted ex vivo proliferation in both neonatal and adult cardiomyocytes isolated from rats. This proposal builds on data from the applicants, an established team from SBP (Drs. Larson and Colas) with basic biology and drug discovery expertise in the field and access to all necessary technologies. The overall goal of this proposal is to generate chemical biology research tools and the starting points for new drugs. As the critical path assays are all in place, we anticipate we can rapidly obtain such probe molecules and start to explore their activity. Our future plans beyond this grant are to ultimately determine their suitability for hit-to-lead activities, begin in vivo evaluation of lead compounds in animal models and eventually patients, and determine their cellular mechanism of action. The product of the work proposed in this application will serve as preliminary data for both hit-to-lead (HTL) grant submissions and parent R01 grant submissions to pursue basic biology understanding of their mechanisms.